Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices

Abstract

A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075 ng/5 μL injection). As confirmed by UHPLC-MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD<5%). A nutritionally relevant amount of active vitamin B12 was produced by P. freudenreichii in cereal malt matrices (up to 1.9 μg/100 g) in 24h at 28 °C.

In situ production of active vitamin B12 in cereal matrices using Propionibacterium freudenreichii

“…The B12 content of the fermented cereal matrices was determined after extraction as cyanocobalamin by the UHPLC–UV and MBA methods as previously reported (Chamlagain et al., ). The identification was confirmed by analyzing the extracts with a high‐resolution mass spectrometer.…”

“…The absorption spectra were recorded, and a chromatogram at 361 nm was acquired. A gradient flow of MilliQ‐water and acetonitrile (both modified with 0.025% TFA) was run for 10 min (Chamlagain et al., ). The B12 concentration of the samples was calculated, using an external calibration curve obtained by injecting cyanocobalamin standards (0.04–0.4 mg·L −1 ).…”

“…The use of P. freudenreichii in cereal‐based products (e.g., bread) is currently limited to improving the product shelf life (Suomalainen & Mäyrä‐Makinen, ; Tinzl‐Malang, Rast, Grattepanche, Sych, & Lacroix, ). However, our recent studies indicated the organisms' abilities to produce active B12 in cereal‐based matrices (Chamlagain et al., ; Edelmann, Chamlagain, Santin, Kariluoto, & Piironen, ). In P. freudenreichii, the availability of cobalt and the B12 lower ligand DMBI was shown to be critical to improving active B12 production (Hugenschmidt, Schwenninger, & Lacroix, ).…”

Food-Like Growth Conditions Support Production of Active Vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the Lower Ligand Base, or Cobalt Supplementation

“…The cobalamin (vitamin B12) and its analog form (pseudovitamin B12) in cells was analyzed as previously described (Chamlagain et al, 2015; Deptula et al, 2015). Briefly, the wet-cell mass from equal volumes of cultures was weighed and suspended with 10 mL of extraction buffer (pH 4.5, 8.3 mM sodium hydroxide, and 20.7 mM acetic acid).…”

Data Intensive Study of Accessibility of Edible Species and Healthcare Across the Globe

“…65,66) . So fermented foods may contribute to brain health via direct and indirect pathways because fermentation may amplify the specific nutrient or phytonutrient content of foods, the ultimate value of which is associated with mental health 67) .…”